Faculty of Applied Sciences
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Item Laccases as biocatalysts for the biosynthesis of hybrid antioxidants(2021) Ngubane, Sandile; Kudanga, Tukayi; Permaul, KugenThe past century has seen an overwhelming upsurge in research interest concerning natural antioxidants, primarily due to rising awareness and knowledge regarding the carcinogenicity of the previously used synthetic antioxidants. Owing to their functional role in numerous redox systems, natural phenolic antioxidants can be applied in diverse areas such as pharmaceuticals, food products, dietary supplements, cosmetics and many other products. However, some natural antioxidants have been shown to exhibit undesirable properties such as low solubility (leads to instability in certain solvents), low bioavailability, low heat stability, low antioxidant capacity and pro-oxidant activity when present at high concentrations alongside transition metal ions such as Cu2+ and Fe3+. Structural modification of these natural compounds is accomplished by chemical or enzymatic means. Biocatalysis has attracted notable attention as a viable way to modify and synthesise bioactive compounds. Laccases are better suited for this function since they can be applied in a plethora of environmentally benign organic synthesis mechanisms through bond formation reactions such as oxidative decomposition, nuclear amination, thio bond formation, oxidative coupling, and C-C bond forming reactions. In this study, the biotransformation of natural phenolic compounds using laccases from Trametes pubescens CBS 696.94 was investigated. Before its application, laccase was biochemically characterised and had its thermodynamic parameters determined. Catechol, gallic acid, quercetin and nordihydroguaiaretic acid were identified as promising substrates and were used in subsequent hetero-coupling studies. Hetero-coupling reactions were carried out in a mixture of a water-miscible and a buffer. Products were monitored using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), purified using preparative TLC and column chromatography, and their molecular weight determined using liquid chromatography-mass spectrometry (LCMS). The antioxidant activities of the products were determined by using the ferric reducing antioxidant power (FRAP), 2,2- diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging assays. The antibacterial activities of the products were assessed against selected American Type Culture Collection (ATCC) bacteria, and their minimum inhibitory concentrations (MICs) were determined. T. pubescens CBS 696.94 produced high titres of extracellular laccase (2330 ± 50 U/l). The enzyme (~58 kDa) had an optimum activity at 60°C while optimum pH varied with the substrate used. The activity was shown to drop drastically at temperatures above 60°C, while the enzyme was most stable between pH 4.5 and 5.0. Enzyme activity was enhanced when the enzyme was pre-incubated in 20 mM CuSO4. The kinetic constants (Km) values for ABTS, syringaldazine (SGZ), 2,6-dimethoxy phenol (2,6-DMP) and guaiacol were 198 µM, 211 µM, 168 µM, and 102 µM, respectively. The kcat values were 103 s-1, 32 s-1, 12 s-1, and 13 s-1 with corresponding catalytic efficiency values (kcat/ Km) of 5.2×105 s-1 M-1, 5.8×104 s1 M-1, 1.9×105 s-1 M-1 and 1.2×105 s-1 M-1, respectively. The t1/2 values of the T. pubescens CBS 696.94 laccase at 50°C, 60°C and 70°C were 7.8 h, 3.8 h, and 0.72 h, respectively. The enzyme deactivation energy (Ed) was 109.362 kJ/mol while ΔG, ΔH, and ΔS for thermal inactivation of the T. pubescens CBS 696.94 laccase were all positive. The enzyme was susceptible to non-competitive (in the presence of sodium azide and sodium dodecyl sulphate) or uncompetitive modes of inhibition (in the presence of L-cysteine, hydrogen peroxide and dithiothreitol). Three heterodimers (catechol + quercetin, quercetin + nordihydroguaiaretic acid (NDGA) and gallic acid + nordihydroguaiaretic acid) and a single heterotrimer (2× quercetin and 1× catechol) were successfully produced, purified and partially characterised. The large scale catechol/quercetin coupling reaction yielded 15.6 ± 1.26% and 9.8 ± 1.12% of the heterodimer and heterotrimer, respectively. The best yields of the catechol/quercetin product were achieved in a monophasic system consisting of 50% dioxane and sodium acetate buffer pH 5.0, with shaking at 200 rpm, temperature 37°C and reaction time 6 h. The products heterodimer showed inferior antioxidant activity, while the heterotrimer displayed enhanced antimicrobial activity against Listeria monocytogenes and Staphylococcus aureus at minimum inhibitory concentrations of 200 and 150 µg/ml, respectively. Large scale reaction of the quercetin/NDGA coupling reaction yielded 14.71 ± 0.59% of the heterodimer. The optimum yield was achieved in a monophasic system consisting of 50% dioxane and sodium acetate buffer pH 5.0, with shaking at 200 rpm, temperature 37°C and reaction time 6 h. The heterodimer showed superior antioxidant activity, exhibiting 1.3 and 1.9-fold increases in the ABTS radical scavenging capacity, 1.3- and 2.0-fold increases in DPPH radical scavenging activity, and 1.14- and 1.6-fold increases in FRAP units when compared to quercetin and nordihydroguaiaretic acid, respectively. It also showed enhanced antimicrobial activity against L. monocytogenes, S. aureus, Escherichia coli and Enterobacter cloacae at minimum inhibitory concentrations of 200, 100 and 50 µg/ml. The large scale reaction of the gallic acid/NDGA coupling reaction yielded 14.12 ± 0.53% of the heterodimer. The optimum yield was achieved in a monophasic system consisting of 60% dioxane and sodium acetate buffer pH 5.0, with shaking at 200 rpm, temperature 37°C and reaction time 6 h. The heterodimer displayed superior antioxidant activity, exhibiting 1.7- and 2.2-fold increases in the ABTS radical scavenging capacity, 2.1- and 3.0-fold increases in DPPH radical scavenging activity, and 1.4- and 1.8-fold increases in FRAP units when compared to nordihydroguaiaretic acid and gallic acid, respectively. In conclusion, two antioxidant and antibacterial compounds were successfully produced, purified and characterised. Overall, this study has demonstrated that laccases from T. pubescens CBS 696.94 can facilitate the cross-coupling of phenolic compounds to form hybrid compounds with enhanced antioxidant and antibacterial activity.Item Extraction and purification of C-phycocyanin and genome analysis of an indigenous hypersaline cyanobacterium(2020) Mogany, Trisha; Bux, Faizal; Swalaha, Feroz Mahomed; Pillai, Sheena Kumari KuttanCyanobacteria are photosynthetic microorganisms that inhabit diverse ecological habitats and are capable of producing wide range of natural products and bioactive metabolities including peptides, vitamins, enzymes and pigments such as phycobiliproteins. Amongst the group of phycobiliproteins, C-Phycocyanin (C-PC) is a light-harvesting accessory pigment known to possess excellent biotechnological applications due to their intense colour, fluorescent properties and health benefits. This study has focused on the characterisation and full genome analysis of a unique indigenous halophilic cyanobacterium capable of overproducing the pigment phycocyanin (C-PC). Further, development of a cost-effective extraction method for high purity C-PC and characterisation of the purified C-PC was accomplished. The strain was isolated from a hypersaline environment in KwaZulu-Natal, South Africa and was found to possess several unique traits such as its ability to accumulate high amount of phycocyanin, tolerance to high salinity (up to 180 g/L), ability to grow under varying growth conditions and high growth rate. The taxonomic identity of the isolate was revealed using a polyphasic approach including cell morphology, growth conditions, pigment composition, 16S rRNA analysis. The cells were oval to rod-shaped, 14-18 μm in size, and contained majority of C-PC, as well as some allophycocyanin and chlorophyll. The strain was moderately thermotolerant (35°C), alkalitolerant (pH 8.5) and was halophilic with an optimum NaCl of 120 g/L. Based on the 16S rRNA gene sequence phylogeny, the strain was found to be related to members of the ‘Euhalothece’ subcluster (99%). Further, the whole genome sequence was also determined, and the annotated genes have shown sequence similarity (90%) to the gas- vacuolate, spindle-shaped Dactylococcopsis salina PCC 8305. Based on the above results, the strain is considered to represent a novel species of Euhalothece. The size of the genome was determined to be 5,113,178 bp and contained 4332 protein-coding genes and 69 RNA genes with a GC content of 46.7%. The full genome sequence analysis also provided important information about the strain which facilitated the identification of key genes and proteins necessary for C-PC synthesis and salt acclimation. Genes encoding osmoregulation, oxidative stress, heat shock, persister cells, and UV-absorbing secondary metabolites, among others, were also identified. Further, single factor experiments were performed to optimise the factors (extraction buffers, freezing time, biomass:buffer ratio and lysozyme concentration) essential for C-PC extraction from cyanobacteria. A range of buffers viz., acetate, potassium phosphate (PPB), sodium phosphate (SPB), phosphate buffered saline (PPBS), Tris-chloride and double distilled water (control) with different pH and concentrations were investigated. Cell lysis was carried out by freezing the cells at different temperatures viz., at -196, and -80, and -20°C, and by thawing at 4 and 25°C. The freezing and thawing time varied from 0.5-24 h. Based on the results obtained, thawing temperature, enzyme concentration and biomass-buffer ratio were further selected for optimisation for maximum C-PC yield and purity using response surface methodology (RSM). Under optimised conditions, the yield of crude C-PC was increased to 78 mg/g (>90% percentage increase) with a purity index of 2.5 compared to extraction prior to optimisation. The crude C-PC was further purified using 6% w/v of activated charcoal combined with a two-step ammonium sulphate (NH4SO4) precipitation and ultrafiltration which resulted in high yield analytical grade C-PC with a purity index of 5. The purified C-PC showed a single absorption peak at 620 nm and emission at 640 nm. Based on the amino acid analysis the calculated molecular weight of α- and β-subunits were found to be 17.7 and 18.4 kDa respectively, which corresponded to the two bands seen on the SDS- PAGE. Additionally, the primary, secondary and tertiary structures of the C-PC was also evaluated based on the amino acid sequence obtained from the genome sequence. The C-PC physiochemical properties such as the molecular weight, isoelectric point, extinction coefficient, half-life, aliphatic index, amino acid property, instability index and Grand Average of Hydropathicity was predicted based on the in-silico analysis of the amino acid sequences. The physicochemical properties revealed that these proteins are non-polar and stable. Multiple sequence alignment analyses of the α- and β-subunits displayed significant differences amongst the amino acid residues of hypersaline/marine and freshwater cyanobacteria. These amino acids play a vital role in the stability of the C-PC. The secondary structure prediction of the α- and β -subunits consisted of > 50% of amino acid residues in α-helices, with 9-13% of amino acid residues in the extended strand. The stability of the purified C-PC under different conditions were investigated. The optimum pH range for purified C-PC was found to be 5.0–7.0 and was found to be stable up to 45oC. However, the relative concentration C-PC (CR%) and thermostability of the purified C-PC was observed to be pH dependent, a lower pH improved the stability at higher temperatures and vice-versa. An IC50 value of 0.540 ± 0.02 mg/mL was also observed using the DPPH assay indicating a higher antioxidant potential of the C-PC. C-phycocyanin exhibited a maximum absorbance of 1.37 ± 0.05 by ferric ion reducing assay. The presence of a high level of non-polar and aromatic residues such as Ala, Gly Glu, Leu, Arg, Ser, and Val could be regarded as an indication of higher antioxidant activity levels of the C-PC. Addition of preservatives sodium azide and sodium citrate (at 4°C) proved to be suitable for preservation of C-PC for up to 42 weeks. This research contributed to our understanding of molecular, cellular and biochemical mechanisms of the C-PC biosynthesis as well as newly identified metabolites in cyanobacteria. The study has also demonstrated an efficient extraction method for analytical grade C-PC from cyanobacterial strains for potential applications in biotechnological biomedical industries.Item Biosynthesis of ZnO nanoparticles using Jacaranda mimosifolia flowers extract: Synergistic antibacterial activity and molecular simulated facet specific adsorption studies(Elsevier, 2016) Sharma, Deepali; Sabela, Myalowenkosi Innocent; Kanchi, Suvardhan; Mdluli, Phumlani Selby; Singh, Gulshan; Stenström, Thor-Axel; Bisetty, KrishnaThe naturally occurring biomolecules present in the plant extracts have been identified to play an active role in the single step formation of nanoparticles with varied morphologies and sizes which is greener and environmen-tally benign. In the present work, spherical zinc oxide nanoparticles (ZnO NPs) of 2–4 nm size were synthesized using aqueous extract of fallen Jacaranda mimosifolia flowers (JMFs), treated as waste. The microwave assisted synthesis was completed successfully within 5 min. Thereafter, phase identification, morphology and optical band gap of the synthesized ZnO NPs were done using X-ray diffraction (XRD), high resolution transmission elec-tron microscopy (HRTEM) and UV–Visible spectroscopy techniques. The composition of JMFs extract was ana-lyzed by gas chromatography–mass spectrometry (GC–MS) and the ZnO NPs confirmation was further explored with fourier transform infrared spectroscopy (FTIR). The GC–MS results confirmed the presence of oleic acid which has high propensity of acting as a reducing and capping agent. The UV–Visible data suggested an optical band gap of 4.03 eV for ZnO NPs indicating their small size due to quantum confinement. Further, facet specific adsorption of oleic acid on the surface of ZnO NPs was studied computationally to find out the im-pact of biomolecules in defining the shape and size of NPs. The viability of gram negative Escherichia coli and gram positive Enterococcus faecium bacteria was found to be 48% and 43%, respectively at high concentration of NPs.